Cloning, Overexpression and Purification of Bacillus subtilis Elongation Factor Tu in Escherichia coli

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His₆ tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.     

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.     

Application of LFH-PCR for the disruption ofSpoIIIE andSpoIIIG ofB. subtilis

The application of LFH-PCR (long flanking homology region-PCR) forBacillus subtilis gene disruption is presented. Without plasmid- or phage-vector construction, only by PCR, based on a DNA sequence retrieved fromB. subtilis genome data base, kanamycin resistance gene was inserted into two genes ofB. subtilis involved in sporulation,spoIIIE andspoIIIG. The effect of gene disruption on subtilisin expression was examined and the sporulation frequency of two mutants was compared to that of the host strain. For this purpose, only 2 or 3 rounds of PCR were required with 4 primers. We first demonstrated the possibility of LFH-PCR for rapid gene disruption to characterize an unknown functional gene ofB. subtilis or other prokaryote in the genomic era.     

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.     

Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Highly bioluminescent Bacillus subtilis obtained through high-level expression of a luxAB fusion gene.

Summary Bioluminescence levels comparable to those achievable in Escherichia coli have yet to be obtained from luxAB expression in gram-positive bacteria. In this communication we describe the gene engineering required to generate a highly bioluminescent derivative of Bacillus subtilis. The combination of a powerful promoter, P _xyn , a fusion derivative of luxAB from Vibrio harveyi and translational coupling have overcome the previously reported limitations in luxAB expression. The implications for highly bioluminescent gram-positive organisms are discussed.     

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

Peroxynitrite scavenging by ferrous truncated hemoglobin GlbO from Mycobacterium leprae

Mycobacterium leprae GlbO has been proposed to represent merging of both O(2) uptake/transport and scavenging of nitrogen reactive species. Peroxynitrite reacts with M. leprae GlbO(II)-NO leading to GlbO(III) via the GlbO(III)-NO species. The value of the second order rate constant for GlbO(III)-NO formation is >1x10(8)M(-1)s(-1) in the absence and presence of CO(2) (1.2x10(-3)M). The CO(2)-independent value of the first order rate constant for GlbO(III)-NO denitrosylation is (2.5+/-0.4)x10(1)s(-1). Furthermore, peroxynitrite reacts with GlbO(II)-O(2) leading to GlbO(III) via the GlbO(IV)O species. Values of the second order rate constant for GlbO(IV)O formation are (4.8+/-0.5)x10(4) and (6.3+/-0.7)x10(5)M(-1)s(-1) in the absence and presence of CO(2) (=1.2x10(-3)M), respectively. The value of the second order rate constant for the peroxynitrite-mediated GlbO(IV)O reduction (= (1.5+/-0.2)x10(4)M(-1)s(-1)) is CO(2)-independent. These data argue for a role of GlbO in the defense of M. leprae against nitrosative stress.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Repeated use of Bacillus subtilis cell walls for copper binding

Purified cell walls from Bacillus subtilis were repeatedly suspended in 5 mm CuCl₂ and, after removing unbound Cu, were suspended in 1% (v/v) HNO₃ to release bound Cu. The walls were then regenerated by washing in H₂O. After five cycles, copper binding actually increased slightly, probably due to enhanced exposure of binding sites in the walls. Thus bacterial walls may be used repeatedly for metal removal during bioremediation of heavy metal pollution.R.J.C. McLean is with the Department of Biology, Southwest Texas State University, 601 University Drive, San Marcos, TX 78666-4616, USA A.M. Campbell is with the Department of Chemical Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. P.T. Khu is with the Department of Mining Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. A.T. Persaud, L.E. Bickerton and D. Beauchemin are with the Department of Chemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada.     

Development and Application of a Novel Signal Peptide Probe Vector with PGA as Reporter in Bacillus subtilis WB700: Twenty-Four Tat Pathway Signal Peptides from Bacillus subtilis were Monitored

In this study, we have developed a novel, versatile signal peptide probe vector driven by promoter P43 in Bacillus subtilis WB700, using Penicillin G Acylase (PGA) as reporter. Twenty-four signal peptides considered belonging to twin-arginine translocation (Tat) pathway were cloned into the probe vector to direct the secretion expression of PGA, respectively. Through 6-nitro-3-phenylacetamidobenzoic acid (NIPAB) filter paper assay, four signal peptides (AmyX, AlbB, LipA, and YmzC) were chosen for further investigation. The extracellular production of PGA demonstrated that these recombinants mediated efficient secretion expression in B. subtilis WB700, in which the maximum activity reached 0.11, 0.21, 0.08, and 0.26 U/mL, respectively. Thus, we provided an efficient tool for easy detection of the signal peptides in B. subtilis, and demonstrated the efficiency of Tat pathway signal peptides via PGA secretion in B. subtilis WB700.     

Cloning and expression in Escherichia coli, Bacillus subtilis, and Streptococcus sanguis of a gene for staphylokinase--a bacterial plasminogen activator

Summary The gene coding for the bacterial plasminogen activator staphylokinase was cloned from the Staphylococcus aureus phage 42D, a serogroup F phage used for lysotyping, onto the standard Escherichia coli plasmid vector pACYC184. The coding and flanking sequences of the sak42D gene were largely identical to those of a sak gene cloned from the serologically different S. aureus phage SøC (Sako and Tsuchida 1983). Subcloning of a 2.5 kb phage 42D DNA fragment onto plasmid pGB3631 allowed the sak42D gene to be introduced into the gram-positive hosts Bacillus subtilis and Streptococcus sanguis. The sak42D gene was expressed and secreted most efficiently by B. subtilis cells (25 g/ml of culture supernatant) reduced in exoprotease production. In this host expression and secretion of Sak was initiated at the early growth phase and continued through the logarithmic phase. Formation of Sak was, however, also observed with the other cloning hosts. The Sak elaborated by the heterologous hosts was serologically identical with authentic Sak derived from S. aureus.     

Nonpungent Capsicum fermentation by Bacillus subtilis and the addition of Rapidase

To reduce the pungency of Capsicum without the loss of its biological activity, a Capsicum sp. was fermented by Bacillus subtilis with the addition of Rapidase enzyme. At 1 day of fermentation, the capsaicin content of the Capsicum ferment with Rapidase had sharply decreased from an initial content of 11.8 to 2.8 μg/ml. The Rapidase-fermented Capsicum had higher total flavonoid and phenolic contents than the Capsicum ferment without Rapidase. In addition, ABTS radical scavenging activity was enhanced in the Rapidase-fermented Capsicum as compared to that without Rapidase. Overall, fermentation using B. subtilis and Rapidase was an efficient method to produce a non-pungent Capsicum with antioxidant properties.     

Spectroscopic characterization of a truncated hemoglobin from the nitrogen-fixing bacterium Herbaspirillum seropedicae

The Herbaspirillum seropedicae genome sequence encodes a truncated hemoglobin typical of group II (Hs-trHb1) members of this family. We show that His-tagged recombinant Hs-trHb1 is monomeric in solution, and its optical spectrum resembles those of previously reported globins. NMR analysis allowed us to assign heme substituents. All data suggest that Hs-trHb1 undergoes a transition from an aquomet form in the ferric state to a hexacoordinate low-spin form in the ferrous state. The close positions of Ser-E7, Lys-E10, Tyr-B10, and His-CD1 in the distal pocket place them as candidates for heme coordination and ligand regulation. Peroxide degradation kinetics suggests an easy access to the heme pocket, as the protein offered no protection against peroxide degradation when compared with free heme. The high solvent exposure of the heme may be due to the presence of a flexible loop in the access pocket, as suggested by a structural model obtained by using homologous globins as templates. The truncated hemoglobin described here has unique features among truncated hemoglobins and may function in the facilitation of O₂ transfer and scavenging, playing an important role in the nitrogen-fixation mechanism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.